This R01 application entitled Lead optimization of novel ML-IAP antagonists to treat lung cancer is in response to PAR-14-279 Discovery of in vivo Chemical Probes. Disruption of normal cell death processes is a hallmark of cancer leading to escape of tumorigenic cells from apoptotic stimuli as well as substantially increased resistance to chemo- and radiation therapies. Inhibitor of Apoptosis Proteins (IAPs) are a family of proteins that regulate cell death and are characterized by a common structural feature, the baculovirus IAP repeat (BIR) domain. The IAPs regulate cell death processes in multiple ways including inhibition of apoptosis/necrosis, and the regulation of cell cycle and inflammation. IAPs in turn are modulated by SMAC (a.k.a. Diablo), a mitochondrial protein that binds directly to the BIR domains. Numerous studies have identified IAPs as potential therapeutic targets for the treatment of cancer. One member of the IAP family, ML-IAP, stands out as a viable target. This is supported by studies on its function within the apoptotic signaling network as well as its role as a biomarker for disease prognosis. Furthermore, ML-IAP has been identified as an attractive target in lung cancer. Inhibition of ML-IAP in this malignancy leads to a substantial reduction in tumor growth as well as sensitization to traditional standard of care therapies. Almost all studies on ML-IAP to date were based on gene ablation studies using RNA interference, because no selective ML-IAP antagonists were available. We recently reported a series of highly potent and selective ML-IAP antagonists, which were generated utilizing a rational design approach mimicking the SMAC-IAP interaction. The most advanced compounds from this series are potent and highly selective inhibitors of ML-IAP in vitro, blocks resistance to chemotherapeutics in whole cells, halts tumor cell proliferation, are non-toxic in normal cells, and show promising drug levels in mice following a single systemic dose (10 mg/kg i.p.). However, the drug-like attributes of the lead compounds (including the pharmacokinetic properties) must be improved, while retaining or enhancing potency and selectivity, to provide compounds suitable for chronic dosing in rodents. We are now poised to initiate full-scale chemistry optimization to provide lead compounds ready for in vivo proof-of-concept studies. Therefore our Specific Aims are: 1. Design and synthesize optimized ML-IAP inhibitors that are orally active in vivo. 2. Assess the potency and selectivity of ML-IAP inhibitors in relevant in vitro assays. 3. Evaluate novel small molecule ML-IAP inhibitors using in vitro ADME/T and in vivo pharmacokinetic (PK) assays. 4. Determine efficacy of lead ML-IAP inhibitor probes in relevant mouse tumorigenic (xenograft) models of lung cancer. The ML-IAP inhibitors generated will provide powerful tools for testing the hypothesis that inhibition of ML-IAP is an effective method for killing tumor cells, while layig a foundation for future development of a novel class of medications for the treatment of lung cancer.